Project Summary Riboswitches are important and ubiquitous regulatory elements that bind small-molecule effectors and control gene expression. Many of the known riboswitch effectors have been identified based in part on the genes under riboswitch control. However, in some cases this flow of information has been reversed, where the identification of a riboswitch class has provided valuable insight about the function of the genes under its regulation. This proposal describes the search for the ligands for new classes of riboswitches, their structural characterization, and the molecular basis of altered specificity. We will focus on prominent RNA motifs for which no ligand has been identified. We will determine the crystal structures of these RNAs. We will determine which nucleotides are necessary for altered specificity and function for several riboswitches that have variant subclasses. More than 50 ?orphan? riboswitch candidates do not have validated ligands. Some of these orphan riboswitch candidates present enormous opportunities to expand our knowledge of important metabolic and signaling processes in species from various domains of life. We will pursue the identification of the ligands for the most prominent orphan riboswitch candidates. To increase the probability that our hypotheses regarding the ligand identity for an orphan riboswitch is strong for the largest number of orphan candidates, we will employ a new genetic screening/selection approach to link the function of a riboswitch to additional genes in a surrogate microorganism. We will pursue crystallization of multiple riboswitches as they are identified. The structures will establish the mechanism of ligand binding and further reveal the complexity of RNA tertiary folds. Structural analysis of ligand binding and investigation of helical switching will allow for feedback into riboswitch search methods, facilitating discovery of previously overlooked candidates. Furthermore, each structure will provide insights that will inform the search for riboswitch subclasses. There are several examples where small changes in riboswitch sequence result in dramatic changes to the specificity of the ligand. We will pursue studies of three riboswitch families that have variant subclasses. We have developed a massively parallel sequencing-based approach to generate quantitative riboswitch activity profiles for thousands of individual mutants simultaneously. This work will establish evolutionary pathways for RNA function and the energetic landscape of substrate specificity and promiscuity. It will also aid in discovery of new subclasses and therefore new biology.